We reported previously that a large fraction of melanoma cell lines induced a suboptimal activation of specific CTL clones, characterized by good tumor cell lysis but no detectable IL-2 production. Using synthetic peptides, we demonstrated recently that this was due to expression of subthreshold levels of appropriate MHC-peptide complexes. We measure here by semiquan-titative reverse transcription-PCR the expression of two melanoma Ag (NA17-A and Melan-A/MART-I) mRNAs in 13 melanoma cell lines and analyze the responses to these cell lines of specific HLA-A2-restricted CTL clones. In line with the idea that the density of MHC-antigenic peptide complexes on melanoma cells is a direct function of the Ag's mRNA level, we found that CTL lysis was grossly proportional to this level. We also established that a minimal level of transcription is required for melanoma cells to induce IL-2 secretion. Interestingly, all cell lines that expressed the Ag above this minimal level, either spontaneously or after gene transfection, stimulated the secretion by tumor-infiltrating lymphocyte of IL-2 amounts proportional to Ag expression unless they exhibited a defective expression of intracellular adhesion molecule-1 or LFA-3 molecules or a low expression of the restricting HLA element. These results indicate that optimal activation and therefore, doubtless, full functionality of melanoma-specific CTL clones critically depend on the mRNA level of the Ag in tumor cells and also on a minimal expression of the HLA restriction element, intracellular adhesion molecule-I, and LFA-3. These data provide a rationale for a better selection of patients to be included in Ag-specific immunization protocols.